Mutant having uracil phosphoribosyl transferase activity

ABSTRACT

The focus of the present invention is a polypeptide having a uracil phosphoribosyl transferase (UPRTase) activity achieved by mutation of one or more residues of the UPRTase. The invention also relates to a nucleotide sequence coding for the UPRTase mutant, a vector for expressing the nucleotide sequence, a viral particle, and a host cell, as well as a composition containing these. The invention further relates to the therapeutic use and method of treatment by using the mutant UPRTases and related compositions. The invention is particularly useful in the context of therapy by suicide genes, notably for treating proliferative and infectious diseases.

The present invention relates to a polypeptide which possesses uracilphosphoribosyl transferase (UPRTase) activity and which is derived froma native UPRTase by one or more residues of said UPRTase being mutated.The invention also relates to a nucleotide sequence which encodes thismutant UPRTase, to a vector for expressing this latter mutant, to aviral particle and a host cell, and to a composition which comprisesthem. Finally, the invention is also directed towards their therapeuticuse and to a method of treatment which implements them. The presentinvention is particularly useful, within the context of suicide genetherapy, for an application with respect, in particular, toproliferative and infectious diseases.

Gene therapy is defined as being the transfer of genetic informationinto a host cell or organism. The first protocol applied to man wasinitiated in the United States, in September 1990, on a patient who wasgenetically immunodeficient on account of a mutation which affected thegene encoding Adenine Deaminase (ADA). The relative success of thisfirst experiment encouraged the development of this approach for avariety of diseases, including both genetic diseases (with the aim ofcorrecting the malfunction of a defective gene) and acquired diseases(cancers, infectious diseases, such as AIDS, etc.). This technology hasexperienced a large number of developments since then, including“suicide gene” therapy, which uses genes whose expression products areable to transform an inactive substance (prodrug) into a cytotoxicsubstance, thereby giving rise to cell death. In 1992, several groupsdemonstrated the relevance of this novel approach for treating tumorsand inhibiting dissemination of the HIV virus, which is responsible forAIDS.

In this respect, the gene encoding the herpes simplex type 1 virusthymidine kinase (HSV-1 TK) constitutes the prototype of the suicidegenes (Caruso et al., 1993, Proc. Natl. Acad. Sci. USA 90, 7024-7028;Culver et al., 1992, Science 256, 1550-1552; Ram et al., 1997, Nat. Med.3, 1354-1361). While the TK polypeptide is not toxic as such, itcatalyzes the transformation of nucleoside analogues such as acycloviror ganciclovir (GCV). The modified nucleosides are incorporated into theDNA chains which are in the process of elongation, inhibiting celldivision as a consequence. A large number of suicide gene/prodrug pairsare currently available. Those which may more specifically be mentionedare rat cytochrome p450 and cyclophosphophamide [sic] (Wei et al., 1994,Human Gene Therapy 5, 969-978), Escherichia coli (E. Coli) purinenucleoside phosphorylase and 6-methylpurine deoxyribonucleoside(Sorscher et al., 1994, Gene Therapy 1, 223-238), E. coli guaninephosphoribosyl transferase and 6-thioxanthine (Mzoz and Moolten, 1993,Human Gene Therapy 4, 589-595) and cytosine deaminase (CDase) and5-fluorocytosine (5FC).

CDase is involved in the pyrimidine metabolic pathway by which exogenouscytosine is transformed into uracil by means of a hydrolyticdeamination. While CDase activities have been demonstrated inprokaryotes and lower eukaryotes (Jund and Lacroute, 1970, J. Bacteriol.102, 607-615; Beck et al., 1972, J. Bacteriol. 110, 219-228; De Haan etal., 1972, Antonie van Leeuwenhoek 38, 257-263; Hoeprich et al., 1974,J. Inf. Dis. 130, 112-118; Esders and Lynn, 1985, J. Biol. Chem. 260,3915-3922), they are not present in mammals (Koechlin et al., 1966,Biochem Pharmacol. 15, 435-446; Polak et al., 1976, Chemotherapy 22,137-153). The Saccharomyces cerevisiae (S. cerevisiae) FCY1 and the E.coli codA genes, which respectively encode the CDase of these twoorganisms, are known and their sequences have been published (EP 402108; Erbs et al., 1997, Curr. Genet. 31, 1-6; WO93/01281).

CDase also deaminates an analogue of cytosine, i.e. 5-fluorocytosine(5-FC), thereby forming 5-fluorouracil (5-FU), which is a compound whichis highly cytotoxic when it is converted into 5-fluoro-UMP (5-FUMP).Cells which lack CDase activity, either because of a mutation whichinactivates the gene encoding the enzyme or because they are naturallydeficient in this enzyme, as are mammalian cells, are resistant to 5-FC(Jund and Lacroute, 1970, J. Bacteriol, 102, 607-615; Kilstrup et al.,1989, J. Bacteriol. 1989 171, 2124-2127). By contrast, mammalian cellsinto which the sequences encoding CDase activity were transferred becamesensitive to 5-FC (Huber et al., 1993, Cancer Res. 53, 4619-4626; Mullenet al., 1992, Proc. Natl. Acad. Sci. USA 89, 33-37; WO 93/01281). Inaddition, the neighboring, untransformed cells also become sensitive to5-FC (Huber et al., 1994, Proc. Natl. Acad. Sci. USA 91, 8302-8306).This phenomenon, which is termed a bystander effect, is due to the cellswhich are expressing the CDase activity secreting 5-FU, which thenintoxicates the neighboring cells by straightforward diffusion acrossthe plasma membrane. This property of 5-FU in diffusing passivelyrepresents an advantage as compared with the tk/GCV reference system,where the bystander effect requires there to be contact with the cellswhich are expressing tk (Mesnil et al., 1996, Proc. Natl. Acad. Sci. USA93, 1831-1835). All the advantages which CDase offers within the contextof gene therapy, in particular anticancer gene therapy, can therefore bereadily understood.

However, the sensitivity to 5-FC varies a great deal depending on thecell lines employed. For example, a low degree of sensitivity isobserved in human tumor cell lines PANC-1 (pancreatic carcinoma) andSK-BR-3 (breast adenocarcinoma) which have been transduced with aretrovirus expressing the E. coli coda gene (Harris et al., 1994, GeneTherapy 1, 170-175). This undesirable phenomenon could be explained bythe 5-FU which is formed by the enzymic action of the CDase either notbeing converted, or only being converted at a low level, into cytotoxic5-FUMP. This step, which is normally effected in mammalian cells byorotate phosphorybosyl [sic] transferase (Peters et al., 1991, Cancer68, 1903-1909), may be absent in particular tumors and thereby renderthe CDase-based gene therapy inoperative.

In prokaryotes and lower eukaryotes, uracil is transformed into UMP bythe action of uracil phosphoribosyl transferase (UPRTase). This enzymealso converts 5-FU into 5-FUMP. Thus, furl mutants of the yeast S.cerevisiae are resistant to high concentrations of 5-FU (10 mM) and 5-FC(10 mM) because, with there being no UPRTase activity, the 5-FU whicharises from the deamination of the 5-FC by CDase is not transformed intocytotoxic 5-FUMP (Jund and Lacroute, 1970, J. Bacteriol. 102, 607-615).The upp and FUR1 genes, which encode E. coli and S. cerevisiae UPRTase,respectively, have been cloned and sequenced (Andersen et al., 1992,Eur. J. Biochem. 204, 51-56; Kern et al., 1990, Gene 88, 149-157).

In order to remedy these drawbacks, the prior art document WO-A-96/16183recommends using a fusion protein which encodes a two-domain enzymepossessing CDase and UPRTase activities, and demonstrates in vitro thatthe transfer of a hybrid codA::upp or FCY1::FUR1 gene, carried by anexpression plasmid, increases the sensitization of transfected B16 cellsto 5-FC.

The present invention is an improvement of the earlier technique in thatit uses a mutated FUR1 gene which encodes a UPRTase which is deleted inits N-terminal part. The present invention results from the observationthat, after the initiating ATG codon, the FUR1 gene contains a secondATG codon encoding methionine in position 36 of the native protein.

An FUR1 gene has now been constructed which lacks 105 nucleotides at the5′ end of the coding part, thereby making it possible to synthesize aUPRTase from which the first 35 N-terminal residues have been deletedand which starts with the methionine in position 36 in the nativeprotein. It has been shown that the expression product of the mutantgene, designated FUR1Δ105, is able to complement an S. cerevisiae furlmutant, thereby demonstrating that it is functional. Surprisingly, thetruncated mutant exhibits a UPRTase activity which is greater than thatof the native enzyme, as is testified by the enzyme assays which havebeen carried out on COS7 cells transfected with a plasmid expressing thecorresponding gene (FUR1Δ105 gene as compared with the wild-type gene).Three human tumor cell lines, which were selected because of theirresistance to 5-FU, were transduced with the mutant gene carried by anadenoviral vector and exhibit an increased sensitivity to 5-FU in vitro.The sensitivity to 5-FC is also increased if the cells are coinfectedwith adenoviruses which are respectively expressing the FCY1 andFUR1Δ105 genes, as compared with an infection with an adenovirusexpressing FCY1 alone. Even more surprisingly, the fusion protein whichis produced by the hybrid FCY1::FU1Δ105 gene, which results from thein-frame fusion of the FCY1 and truncated FUR1 genes, retains itsUPRTase activity but exhibits a CDase activity which is increased by afactor of 10 to 30 as compared with that measured using the native FCY1product. The high CDase activity of the bifunctional protein makes itpossible to form a pool of 5-FU which generates a substantial bystandereffect. It is to be noted that the CDase activity of the CDase::UPRTasefusion protein of WO-A-96/16183 was not shown to be improved.

The present invention provides a more efficient mutant, thereby makingit possible to increase the sensitivity of cells to 5-FC and to improvethe prospects for gene therapy using suicide genes. This mutant can beused for a large number of applications, in particular anticancer andantiviral applications, and all applications which require cell death.

For this reason, the present invention relates to a polypeptidepossessing a uracil phosphoribosyl transferase (UPRTase) activity,characterized in that it is derived from a native UPRTase at least bymutating one or more residues of said UPRTase.

Within the meaning of the present invention, a polypeptide possessing aUPRTase activity refers to a polypeptide which is able to converturacil, or one of its derivatives, into a monophosphate analog, inparticular 5-FU into 5-FUMP. “Mutation” is to be understood as being theaddition, deletion and/or substitution of one or more residues at anysite in said polypeptide.

The native UPRTase from which the polypeptide according to the inventionis derived can be of any origin, in particular of prokaryotic, fungal oryeast origin. By way of illustration, the UPRTases from E. coli(Anderson et al., 1992, Eur. J. Biochem 204, 51-56), from Lactococcuslactis (Martinussen and Hammer, 1994, J. Bacteriol. 176, 6457-6463),from Mycobacterium bovis (Kim et al., 1997, Biochem Mol. Biol. Int 41,1117-1124) and from Bacillus subtilis (Martinussen et al., 1995, J.Bacteriol. 177, 271-274), can be used within the context of theinvention. However, very particular preference is given to using a yeastUPRTase, in particular that encoded by the S. cerevisiae FUR1 gene,whose sequence is disclosed in Kern et al. (1990, Gene 88, 149-157). Byway of information, the sequences of the genes, and those of thecorresponding UPRTases, can be found in the literature and inspecialized databases (SWISSPROT, EMBL, Genbank, Medline, etc.).

According to one particularly advantageous embodiment, the polypeptideaccording to the invention is a deletion mutant of a native UPRTase. Thedeletion is preferably located in the N-terminal region of the originalUPRTase. The deletion can be total (affecting all the residues of saidN-terminal region) or partial (affecting one or more residues which mayor may not be continuous in the primary structure). In a general manner,a polypeptide consists of an N-terminal part, of a central part and of aC-terminal part, with each part representing approximately one third ofthe molecule. For example, in the case of the S. cerevisiae UPRTase,which contains 251 amino acids, the N-terminal part consists of thefirst 83 residues, starting with the so-called initiating methionine,which is located in the first position of the native form. In the caseof the E. coli UPRTase, the N-terminal part covers positions 1 to 69.

This preferred embodiment can of course be combined with one or moreadditional mutation(s) at any site in the molecule. Preferably, theadditional modification(s) do not significantly affect the UPRTaseenzymic properties of the polypeptide according to the invention. It ispointed out that the biological activity of the mutants can be tested,in particular using the techniques which are described in the exampleswhich follow.

Very preferably, the polypeptide according to the invention is derivedfrom a native UPRTase at least by deleting all or part of the N-terminalregion upstream of the second ATG codon of said native UPRTase. Thetotal deletion of the aforesaid region is preferred. For example, theUPRTase encoded by the FUR1 gene comprises a first ATG codon (initiatingATG codon) in position +1 followed by a second in position +36. Thus, itis possible to envisage deleting residues +1 to 35 within the context ofthe present invention, thereby giving a polypeptide which starts at themethionine which is normally found in position +36 of the native form.

A preferred polypeptide according to the invention comprises an aminoacid sequence which is substantially as depicted in the SID NO: 1sequence identifier, starting at the Met residue in position 1 andfinishing at the Val residue in position 216. The term “substantially”refers to a degree of identity with said SID NO: 1 sequence which isgreater than 70%, advantageously greater than 80%, preferably greaterthan 90% and, very preferably greater than 95%. Still more preferably,the polypeptide comprises the amino acid sequence depicted in the SIDNO: 1 sequence identifier. As mentioned above, it can contain additionalmutations. Substitution of the serine residue at position 2 (position 37in the native UPRTase) with an alanine residue may in particular bementioned.

Advantageously, the polypeptide according to the invention exhibits aUPRTase activity which is appreciably higher than that exhibited by saidnative UPRTase. The results which are presented in the examples whichfollow clearly demonstrate a more rapid and/more efficient conversion of5-FU into 5-FUMP, manifesting itself in a higher degree of cytotoxicitywith regard to the transfected or transduced cells. Advantageously, theUPRTase activity of the polypeptide according to the invention isgreater than that exhibited by the native UPRTase by a factor of from 2to 100, preferably of from 5 to 75, and very preferably of from 10 to50.

According to another embodiment, the polypeptide according to theinvention is a fusion polypeptide in which it is fused in-frame with atleast one second polypeptide. Even though the fusion can take place atany site in the first polypeptide, the N- or C-terminal ends arepreferred, in particular the N-terminal end. Advantageously, thein-frame fusion uses a second polypeptide which exhibits a cytosinedeaminase (CDase) activity and is derived from a native cytosinedeaminase, such that the fusion polypeptide according to the inventionexhibits CDase and UPRTase activities. An FCY1::FUR1 fusion (designatedFCU1 below) is preferred. Such a bifunctional polypeptide makes itpossible to improve the sensitivity of the target cells to 5-FC and5-FU. “Cytosine deaminase activity” is understood as covering thedeamination of cytosine or one of its analogs. Preferably, the secondpolypeptide according to the invention is able to metabolize 5-FC into5-FU.

A CDase of prokaryotic or lower eukaryotic origin is used within thecontext of the present invention. Still more preferably, the CDase is ayeast CDase, in particular that encoded by the Saccharomyces cerevisiaeFCY1 gene. The cloning and the sequence of the genes encoding the CDasesof different origins are available in the literature and the specializeddatabases. For information, the sequence of the FCY1 gene is disclosedin Erbs et al. (1997, Curr. Genet. 31, 1-6). It is of course possible touse a CDase mutant which possesses a conversion ability which iscomparable or superior to that of the native enzyme. The skilled personis capable of cloning the CDase sequences on the basis of the publisheddata, of carrying out any mutations, of testing the enzymic activity ofthe mutant forms in an acellular or cellular system in accordance withthe technique of the art, or following the protocol given below, and offusing the polypeptides having CDase and UPRTase activity in-frame.

A preferred example is a polypeptide which comprises an amino acidsequence which is substantially as depicted in the SID NO: 2 sequenceidentifier, starting at the Met residue in position 1 and finishing atthe Val residue in position 373. The term “substantially” is defined asbefore. A polypeptide which comprises the amino acid sequence asdepicted in the SID NO: 2 sequence identifier is very particularlyappropriate for implementing the invention.

According to an advantageous embodiment, a bifunctional polypeptideaccording to the invention exhibits a CDase activity which isappreciably higher than that of said native CDase. Thus, the exampleswhich follow demonstrate that coupling the two enzymes makes it possibleto increase the sensitization of the target cells to 5-FC. The factor bywhich the sensitization is increased is advantageously at least 2,preferably at least 5 and, very preferably, 10 or more.

In a general manner, a polypeptide according to the invention can beproduced either by the conventional methods of chemical synthesis or byrecombinant DNA techniques (see, for example, Maniatis et al., 1989,Laboratory Manual, Cold Spring Harbor, Laboratory Press, Cold SpringHarbor, N.Y.). For this reason, the present invention also covers apreparation process in which a nucleotide sequence encoding saidpolypeptide is introduced into a cell in order to generate a transformedcell, said transformed cell is cultured under conditions appropriate forenabling said polypeptide to be produced, and said polypeptide isharvested from the cell culture. The producer cell can be of any originand, without limitation, a bacterium, a yeast or a mammalian cell, tothe extent that the nucleotide sequence under consideration is eitherintegrated into its genome or integrated into an appropriate expressionvector which is able to replicate. Naturally, the nucleotide sequence isplaced under the control of transcription and translation signals whichenable it to be expressed in the producer cell. Expression vectors andcontrol signals are known to the skilled person. The polypeptide can berecovered from the medium or the cells (after they have been lyzed) andsubjected to conventional purification steps (by chromatography,electrophoresis, filtration, immunopurification, etc.).

The present invention also relates to a nucleotide sequence whichencodes a polypeptide according to the invention. The nucleotidesequence can be a cDNA or genomic sequence or be of a mixed type. Itcan, where appropriate, contain one or more introns, with these being ofnative, heterologous (for example the intron of the rabbit β-globingene, etc.) or synthetic origin, in order to increase expression in thehost cells. As has already been pointed out, said sequence can encode apolypeptide which is derived from the native enzyme or a mutant whichexhibits a comparable or superior activity. The sequences employedwithin the context of the present invention can be obtained by theconventional techniques of molecular biology, for example by screeninglibraries with specific probes, by immunoscreening expression librariesor by PCR using suitable primers, or by chemical synthesis. The mutantscan be generated from the native sequences by substituting, deletingand/or adding one or more nucleotides using the techniques ofsite-directed mutagenesis, of PCR, of digesting with restriction andligation enzymes, or else by chemical synthesis. The ability of themutants and constructs to function can be verified by assaying theenzymic activity or by measuring the sensitivity of target cells to 5-FCand/or 5-FU.

The present invention also relates to a recombinant vector which carriesa nucleotide sequence according to the invention which is placed underthe control of the elements which are required for expressing it in ahost cell. The recombinant vector can be of plasmid or viral origin andcan, where appropriate, be combined with one or more substances whichimprove the transfectional efficiency and/or stability of the vector.These substances are widely documented in the literature which isavailable to the skilled person (see, for example, Felgner et al., 1987,Proc. West. Pharmacol. Soc. 32, 115-121; Hodgson and Solaiman, 1996,Nature Biotechnology 14, 339-342; Remy et al., 1994, BioconjugateChemistry, 5, 647-654). By way of non-limiting illustration, thesubstances can be polymers, lipids, in particular cationic lipids,liposomes, nuclear proteins or neutral lipids. These substances can beused alone or in combination. A combination which can be envisaged isthat of a recombinant plasmid vector which is combined with cationiclipids (DOGS, DC-CHOL, sperimine-chol, spermidine-chol, etc.) andneutral lipids (DOPE).

The choice of the plasmids which can be used within the context of thepresent invention is immense. They can be cloning vectors and/orexpression vectors. In a general manner, they are known to the skilledperson and, while a number of them are available commercially, it isalso possible to construct them or to modify them using the techniquesof genetic manipulation. Examples which may be mentioned are theplasmids which are derived from pBR322 (Gibco BRL), pUC (Gibco BRL),pBluescript (Stratagene), pREP4, pCEP4 (Invitrogene) or p Poly (Lathe etal., 1987, Gene 57, 193-201). Preferably, a plasmid which is used in thecontext of the present invention contains an origin of replication whichensures that replication is initiated in a producer cell and/or a hostcell (for example, the ColE1 origin will be chosen for a plasmid whichis intended to be produced in E. coli and the oriP/EBNA1 system will bechosen if it desired that the plasmid should be self-replicating in amammalian host cell, Lupton and Levine, 1985, Mol. Cell. Biol. 5,2533-2542; Yates et al., Nature 313, 812-815). The plasmid canadditionally comprise a selection gene which enables the transfectedcells to be selected or identified (complementation of an auxotrophicmutation, gene encoding resistance to an antibiotic, etc.). Naturally,the plasmid can contain additional elements which improve itsmaintenance and/or its stability in a given cell (cer sequence, whichpromotes maintenance of a plasmid in monomeric form (Summers andSherrat, 1984, Cell 36, 1097-1103, sequences for integration into thecell genome).

With regard to a viral vector, it is possible to envisage a vector whichis derived from a poxvirus (vaccinia virus, in particular MVA,canarypoxvirus, etc.), from an adenovirus, from a retrovirus, from aherpesvirus, from an alphavirus, from a foamy virus or from anadenovirus-associated virus. Preference will be given to using a vectorwhich does not replicate and does not integrate. In this respect,adenoviral vectors are very particularly suitable for implementing thepresent invention.

Retroviruses have the property of infecting, and in most casesintegrating into, dividing cells and in this regard are particularlyappropriate for use in relation to cancer. A recombinant retrovirusaccording to the invention generally contains the LTR sequences, anencapsidation region and the nucleotide sequence according to theinvention, which is placed under the control of the retroviral LTR or ofan internal promoter such as those described below. The recombinantretrovirus can be derived from a retrovirus of any origin (murine,primate, feline, human, etc.) and in particular from the MoMuLV (Moloneymurine leukemia virus), MVS (Murine sarcoma virus) or Friend murineretrovirus (Fb29). It is propagated in an encapsidation cell line whichis able to supply in trans the viral polypeptides gag, pol and/or envwhich are required for constituting a viral particle. Such cell linesare described in the literature (PA317, Psi CRIP GP+Am-12 etc.). Theretroviral vector according to the invention can contain modifications,in particular in the LTRs (replacement of the promoter region with aeukaryotic promoter) or the encapsidation region (replacement with aheterologous encapsidation region, for example the VL30 type) (seeFrench applications 94 08300 and 97 05203).

Preference will be given to using an adenoviral vector which lacks allor part of at least one region which is essential for replication andwhich is selected from the E1, E2, E4 and L1-L5 regions in order toavoid the vector being propagated within the host organism or theenvironment. A deletion of the E1 region is preferred. However, it canbe combined with (an)other modification(s)/deletion(s) affecting, inparticular, all or part of the E2, E4 and/or L1-L5 regions, to theextent that the defective essential functions are complemented in transby means of a complementing cell line and/or a helper virus. In thisrespect, it is possible to use second-generation vectors of the state ofthe art (see, for example, international applications WO-A-94/28152 andWO-A-97/04119). By way of illustration, deletion of the major part ofthe E1 region and of the E4 transcription unit is very particularlyadvantageous. For the purpose of increasing the cloning capacities, theadenoviral vector can additionally lack all or part of the non-essentialE3 region. According to another alternative, it is possible to make useof a minimal adenoviral vector which retains the sequences which areessential for encapsidation, namely the 5′ and 3′ ITRs (InvertedTerminal Repeat), and the encapsidation region. The various adenoviralvectors, and the techniques for preparing them, are known (see, forexample, Graham and Prevect, 1991, in Methods in Molecular Biology, Vol7, p 109-128; Ed: E. J. Murey, The Human Press Inc).

Furthermore, the origin of the adenoviral vector according to theinvention can vary both from the point of view of the species and fromthe point of view of the serotype. The vector can be derived from thegenome of an adenovirus of human or animal (canine, avian, bovine,murine, ovine, porcine, simian, etc.) origin or from a hybrid whichcomprises adenoviral genome fragments of at least two different origins.More particular mention may be made of the CAV-1 or CAV-2 adenovirusesof canine origin, of the DAV adenovirus of avian origin or of the Badtype 3 adenovirus of bovine origin (Zakharchuk et al., Arch. Virol.,1993, 128: 171-176; Spibey and Cavanagh, J. Gen. Virol. 1989, 70:165-172; Jouvenne et al., Gene, 1987, 60: 21-28; Mittal et al., J. Gen.Virol., 1995, 76: 93-102). However, preference will be given to anadenoviral vector of human origin which is preferably derived from aserotype C adenovirus, in particular a type 2 or 5 serotype Cadenovirus.

An adenoviral vector according to the present invention can be generatedin vitro in Escherichia coli (E. coli) by ligation or homologousrecombination (see, for example, international applicationWO-A-96/17070) or else by recombination in a complementing cell line.

The elements required for expression consist of all the elements whichenable the nucleotide sequence to be transcribed into RNA and the mRNAto be translated into polypeptide. These elements comprise, inparticular, a promoter which may be regulatable or constitutive.Naturally, the promoter is suited to the chosen vector and the hostcell. Examples which may be mentioned are the eukaryotic promoters ofthe PGK (phosphoglycerate kinase), MT (metallothionein; McIvor et al.,1987, Mol. Cell Biol. 7, 838-848), α-1 antitrypsin, CFTR, surfactant,immunoglobulin, β-actin (Tabin et al., 1982, Mol. Cell Biol. 2, 426-436)and SRα (Takebe et al., 1988, Mol. Cell Biol. 8, 466-472) genes, theearly promoter of the SV40 virus (Simian virus), the LTR of RSV (Roussarcoma virus), the HSV-1 TK promoter, the early promoter of the CMVvirus (Cytomegalovirus), the p7.5K pH5R, pK1L, p28 and p11 promoters ofthe vaccinia virus, and the E1A and MLP adenoviral promoters. Thepromoter can also be a promoter which stimulates expression in a tumoror cancer cell. Particular mention may be made of the promoters of theMUC-1 gene, which is overexpressed in breast and prostate cancers (Chenet al., 1995, J. Clin. Invest. 96, 2775-2782), of the CEA (standing forcarcinoma embryonic antigen) gene, which is overexpressed in coloncancers (Schrewe et al., 1990, Mol. Cell. Biol. 10, 2738-2748) of thetyrosinase gene, which is overexpressed in melanomas (Vile et al., 1993,Cancer Res. 53, 3860-3864), of the ERBB-2 gene, which is overexpressedin breast and pancreatic cancers (Harris et al., 1994, Gene Therapy 1,170-175) and of the α-fetoprotein gene, which is overexpressed in livercancers (Kanai et al., l997, Cancer Res. 57, 461-465). Thecytomegalovirus (CMV) early promoter is very particularly preferred.

The necessary elements can furthermore include additional elements whichimprove the expression of the nucleotide sequence according to theinvention or its maintenance in the host cell. Intron sequences,secretion signal sequences, nuclear localization sequences, internalsites for the reinitiation of translation of IRES type, transcriptiontermination poly A sequences, tripartite leaders and origins ofreplication may in particular be mentioned. These elements are known tothe skilled person.

The recombinant vector according to the invention can also comprise oneor more additional genes of interest, with it being possible for thesegenes to be placed under the control of the same regulatory elements(polycistronic cassette) or of independent elements. Genes which may inparticular be mentioned are the genes encoding interleukins IL-2, IL-4,IL-7, IL-10 and IL-12, interferons, tumor necrosis factor (TNF), colonystimulating factors (CSF), in particular GM-CSF, and factors acting onangiogenesis (for example PAI-1, standing for plasminogen activatorinhibitor). In one particular embodiment, the recombinant vectoraccording to the invention comprises the gene of interest encoding IL-2or encoding interferon γ (INFγ). It is also possible to envisagecombining the nucleotide sequence according to the invention with othersuicide genes such as the HSV-1 TK gene, the ricin gene, the choleratoxin gene, etc.

The present invention also relates to a viral particle which comprises arecombinant vector according to the invention. Such a viral particle canbe generated from a viral vector using any technique which isconventional in the field of the art. The viral particle is propagatedin a complementing cell which is suited to the deficiencies of thevector. With regard to an adenoviral vector, use will, for example, bemade of the 293 cell line, which was established using human embryonickidney cells and which efficiently complements the E1 function (Grahamet al., 1977, J. Gen. Virol. 36, 59-72), of the A549-E1 cell line (Imleret al., 1996, Gene Therapy 3, 75-84) or of a cell line which permitsdouble complementation (Yeh et al., 1996, J. Virol. 70, 559-565;Krougliak and Graham, 1995, Human Gene Therapy 6, 1575-1586; Wang etal., 1995 Gene Therapy 2, 775-783; international application WO97/04119). It is also possible to employ helper viruses to at leastpartially complement the defective functions. A complementing cell isunderstood as being a cell which is able to supply in trans the earlyand/or late factors which are required for encapsidating the viralgenome in a viral capsid in order to generate a viral particle whichcontains the recombinant vector. Said cell may not be able to complementall the defective functions of the vector on its own and, in this case,can be transfected/transduced with a vector/helper virus which suppliesthe additional functions.

The invention also relates to a process for preparing a viral particle,in which process:

(i) a recombinant vector according to the invention is introduced into acomplementing cell which is able to complement said vector in trans, soas to obtain a transfected complementing cell,

(ii) said transfected complementing cell is cultured under conditionswhich are appropriate for enabling said viral particle to be produced,and

(iii) said viral particle is recovered from the cell culture.

While the viral particle can of course be recovered from the culturesupernatant, it can also be recovered from the cells. One of thecommonly employed methods consists in lysing the cells by means ofconsecutive freezing/thawing cycles in order to collect the virions inthe lysis supernatant. The virions can then be amplified and purifiedusing the techniques of the art (chromatographic method, method ofultra-centrifugation, in particular through a cesium chloride gradient,etc.).

The present invention also relates to a host cell which comprises anucleotide sequence or a recombinant vector according to the invention,or is infected with a viral particle according to the invention. For thepurposes of the present invention, a host cell consists of any cellwhich can be transfected with a recombinant vector or can be infectedwith a viral particle, as defined above. A mammalian cell, in particulara human cell is very particularly suitable. The cell can comprise saidvector in a form which is or is not (episome) integrated into thegenome. The cell can be a primary or tumor cell of any origin, inparticular an hematopoietic cell (totipotent stem cell, leukocyte,lymphocyte, monocyte or macrophage, etc.), muscle cell (satellite cell,myocyte, myoblast, smooth muscle cell, etc.), cardiac cell, pulmonarycell, tracheal cell, hepatic cell, epithelial cell or fibroblast.

The present invention also relates to a composition which comprises apolypeptide, a nucleotide sequence, a recombinant vector, a viralparticle or a host cell according to the invention in combination with apharmaceutically acceptable excipient.

The present invention also relates to a composition which comprises apolypeptide according to the invention which exhibits a UPRTase activityand another polypeptide of interest, in particular a polypeptide of theprior art which exhibits a CDase activity.

The present invention furthermore relates to a composition whichcomprises a polypeptide according to the invention and a polypeptide ofinterest which is encoded by one of the previously mentioned genes ofinterest. Of these polypeptides of interest, particular mention may bemade of interleukins IL-2, IL-4, IL-7, IL-10 and IL-12, interferons,tumor necrosis factor (TNF), colony stimulating factors (CSF), inparticular GM-CSF, and factors acting on angiogenesis (for examplePAI-1, standing for plasminogen activator inhibitor). IL-2 or INFγ arevery particularly envisaged.

The composition can also be based on nucleotide sequences which enablethe above polypeptides to be expressed within the host cell. Thenucleotide sequences may be carried by one and the same expressionvector or by two independent vectors. Said composition can of coursecomprise viral particles which are generated from (a) viral vector(s)expressing said nucleotide sequence(s).

For this reason, the present invention also relates to a compositionwhich comprises a nucleotide sequence according to the invention whichencodes a polypeptide exhibiting a URPTase activity and a secondnucleotide sequence of interest which encodes, in particular, apolypeptide exhibiting a CDase activity.

The present invention additionally relates to a composition whichcomprises a nucleotide sequence according to the invention and a secondnucleotide sequence of interest which encodes a polypeptide selectedfrom IL-2 and INFγ.

A composition according to the invention is more specifically intendedfor the preventive or curative treatment of diseases by means of genetherapy and is more specifically aimed at proliferative diseases(cancers, tumors, restenosis, etc.) and at diseases of infectiousorigin, in particular of viral origin (induced by hepatitis B or Cviruses, HIV, herpes, retroviruses, etc.).

A composition according to the invention can be made conventionally witha view to administering it locally, parenterally or by the digestiveroute. In particular, a therapeutically effective quantity of thetherapeutic or prophylactic agent is combined with a pharmaceuticallyacceptable excipient. It is possible to envisage a large number ofroutes of administration. Examples which may be mentioned are theintragastric, subcutaneous, intracardiac, intramuscular, intravenous,intraperitoneal, intratumor, intranasal, intrapulmonary andintratracheal routes. In the case of these three latter embodiments, itis advantageous for administration to take place by means of an aerosolor by means of instillation. The administration can take place as asingle dose or as a dose which is repeated on one or more occasionsafter a particular time interval. The appropriate route ofadministration and dosage vary depending on a variety of parameters, forexample the individual, the disease to be treated or the gene(s) ofinterest to be transferred. The preparations based on viral particlesaccording to the invention can be formulated in the form of doses ofbetween 10⁴ and 10¹⁴ pfu (plaque-forming units), advantageously 10⁵ and10¹³ pfu, preferably 10⁶ and 10¹² pfu. As far as the recombinant vectoraccording to the invention is concerned, it is possible to envisagedoses comprising from 0.01 to 100 mg of DNA, preferably from 0.05 to 10mg, very particularly preferably from 0.5 to 5 mg. A composition basedon polypeptides preferably comprises from 0.05 to 10 g, veryparticularly preferably from 0.05 to 5 g, of said polypeptide.Naturally, the doses can be adjusted by the clinician.

The formulation can also include a diluent, an adjuvant or an excipientwhich is acceptable from the pharmaceutical point of view, as well assolubilizing, stabilizing and preserving agents. In the case of aninjectable administration, preference is given to a formulation in anaqueous, non-aqueous or isotonic solution. It can be presented as asingle dose or as a multidose, in liquid or dry (powder, lyophilizate,etc.) form which can be reconstituted at the time of use using anappropriate diluent. The formulation can also comprise appropriatequantities of prodrugs.

The present invention also relates to the therapeutic or prophylacticuse of a polypeptide, of a recombinant vector, of a viral particle or ofa host cell according to the invention for preparing a medicament whichis intended for treating the human or animal body by gene therapy or byadministering protein which has been produced by the recombinant route.According to a first possibility, the medicament can be administereddirectly in vivo (for example by intravenous injection, into anaccessible tumor, into the lungs by means of an aerosol, into thevascular system using an appropriate catheter, etc.). It is alsopossible to adopt the ex vivo approach, which consists in removing cellsfrom the patient (bone marrow stem cells, peripheral blood lymphocytes,muscle cells, etc.), transfecting or infecting them in vitro inaccordance with the techniques of the art and then readministering themto the patient. A preferred use consists in treating or preventingcancers, tumors and diseases which result from unwanted cellproliferation. Conceivable applications which may be mentioned arecancers of the breast, of the uterus (in particular those induced bypapilloma viruses), of the prostate, of the lung, of the bladder, of theliver, of the colon, of the pancreas, of the stomach, of the esophagus,of the larynx, of the central nervous system and of the blood(lymphomas, leukemia, etc.). It can also be used in the context ofcardiovascular diseases, for example in order to inhibit or retard theproliferation of the smooth muscle cells of the blood vessel wall(restenosis). Finally, in the case of infectious diseases, it ispossible to conceive of the medicament being applied to AIDS.

The invention also extends to a method for treating diseases by genetherapy, characterized in that a nucleotide sequence, a recombinantvector, a viral particle or a host cell according to the invention isadministered to an host organism or cell which is in need of suchtreatment.

When the treatment method makes use of a nucleotide sequence, arecombinant vector or a viral particle enabling a polypeptide accordingto the invention, which possesses a UPRTase activity, to be expressed,it can be advantageous to additionally administer a second nucleotidesequence which encodes a second polypeptide exhibiting a CDase activity,with said second nucleotide sequence being carried by said recombinantvector or viral particle or by an independent vector or viral particle.In this latter case, the UPRTase and CDase sequences can be administeredsimultaneously or consecutively, with the order of administration beingof no importance.

According to an advantageous embodiment, the therapeutic use or thetreatment method also comprises an additional step in whichpharmaceutically acceptable quantities of a prodrug, advantageously ananalog of cytosine, in particular 5-FC, are administered to the hostorganism or cell. By way of illustration, it is possible to use a doseof from 50 to 500 mg/kg/day, with a dose of 200 mg/kg/day beingpreferred. Within the context of the present invention, the prodrug isadministered in accordance with standard practice, with theadministration taking place prior to, concomitantly with or elsesubsequent to the administration of the therapeutic agent according tothe invention. The oral route is preferred. It is possible to administera single dose of prodrug or doses which are repeated for a time which issufficiently long to enable the toxic metabolite to be produced withinthe host organism or cell.

Furthermore, the composition or method according to the invention can becombined with one or more substances which potentiate the cytotoxiceffect of the 5-FU. Mention may in particular be made of drugs whichinhibit the enzymes of the pathway for the de novo biosynthesis of thepyrimidines (for example those mentioned below), drugs such asLeucovorin (Waxman et al., 1982, Eur. J. Cancer Clin. Oncol. 18,685-692), which, in the presence of the product of the metabolism of5-FU (5-FdUMP), increases the inhibition of thymidylate synthase,resulting in a decrease in the pool of dTMP, which is required forreplication, and finally drugs such as methotrexate (Cadman et al.,1979, Science 250, 1135-1137) which, by inhibiting dihydrofolatereductase and increasing the pool of PRPP (phosphoribosylpyrophosphate),brings about an increase in the incorporation of 5-FU into the cellularRNA.

The present invention is also directed towards using the sequences orrecombinant vectors according to the invention as positive selectionmarkers in mammalian cells. Advantageously, the cells are transfectedand the cell mixture is then cultured in the presence of inhibitors ofthe pathway for the de novo biosynthesis of pyrimidines, such as PALA(N-(phosphonoacetyl)-L-aspartate; Moore et al., 1982, Biochem.Pharmacol. 31, 3317-3321), A77 1726 (active metabolite of Leflunomide;Davis et al., 1996, Biochem. 35, 1270-1273) and Brequinar (Chen et al.,1992, Cancer Res. 52, 3251-3257). The presence of such inhibitors blocksthe de novo synthesis of UMP, which is required for synthesizing RNA andDNA, thereby resulting in cell death. This cytotoxic effect can becircumvented by expressing the nucleotide sequence according to theinvention encoding a UPRTase activity in the presence of uracil or bycoexpressing this latter sequence with sequences encoding a CDaseactivity (where appropriate in fused form) in the presence of cytosine.As a consequence, only the transfected cells (cells which haveincorporated the UPRTase/CDase sequences) will be able to grow in thepresence of inhibitors of the pyrimidine synthesis pathway. Thus, theuse according to the invention enables a transfected cell to beefficiently identified in, and/or isolated from, a cell mixture.

The present invention also relates to the use of the sequences orrecombinant vectors according to the invention as negative selectionmarkers in experiments in which the genes of embryonic stem cells, onwhich methods for preparing transgenic animals are based, areinterrupted (knocked out) (see, for example, Capecchi, 1989, Science244, 1288-1292; Reid et al., 1990, Proc. Natl. Acad. Sci. USA 87,4299-4303). Such a use, in combination with the gene for resistance toneomycin, for example, can make it possible to select the cells whichhave undergone an homologous recombination event and which will alone beable to grow in the presence of Geneticin and the correspondingfluorinated pyrimidines (5-FU if a nucleotide sequence according to theinvention is used which encodes a UPRTase activity, and 5-FC when anucleotide sequence encoding a CDase activity is also used). The cellswhich have undergone a non-targeted recombination event are able to growin the presence of Geneticin but not in the presence of the fluorinatedpyrimidines. Another potential use as a negative selection marker is tobe found in the plant field since, just like mammalian cells, plants donot possess any endogenous CDase activity. They can be sensitized to5-FC by transfecting a nucleotide sequence according to the inventionwhich enables an exogenous CDase to be expressed (see, for example,Perera et al. 1993, Plant. Mol. Biol. 23, 797-799).

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is illustrated, without for all that beinglimited, by the following examples in which:

FIG. 1 depicts the rate of survival of B6D2 mice which have beenimplanted with B16F0 tumor cells. The different groups of mice (10 miceper group) are treated with the following compositions:

Vehicle/saline;+Vehicle/5-FC;*Ad null/saline;−Adnull/5-FC;×AdFCU1/saline;♦AdFCU1/5-FC

FIG. 2 depicts the rate of survival of B6D2 mice which have beenimplanted with B16F0 tumor cells. The different groups of mice (10 miceper group) are treated with the following compositions:

Vehicle/saline;+Vehicle/GCV;*Ad null/saline;−Adnull/GCV;×AdTK/saline;♦AdTK/GCV

FIG. 3 depicts the rate of survival of B6D2 mice which have beenimplanted with B16F0 tumor cells. The different groups of mice (13 miceper group) are treated with the following compositions:

Vehicle/saline;+Vehicle/5-FC;*Ad null/saline;−Adnull/5-FC;×Adnull+AdFCU1/saline;♦Adnull+AdFCU1/5-FC;▾Adnull+AdmuIFNg/saline;ΔAdnull+AdmuIFNg/5-FC;AdmuIFNg+AdFCU1/saline;∘AdmuIFNg+AdFCU1/5-FC

FIG. 4 depicts the rate of survival of B6D2 mice which have beenimplanted with B16F0 tumor cells. The different groups of mice (13 miceper group) are treated with the following compositions:

Vehicle/saline;+Vehicle/5-FC;*Ad null/saline;−Adnull/5-FC;×Adnull+AdFCU1/saline; ♦Adnull+AdFCU1/5-FC;▾Adnull+AdhuIL-2/saline;ΔAdnull+AdhuIL-2/5-FC;AdhuIL-2+AdFCU1/saline;∘AdhuIL-2+AdFCU1/5-FC

EXAMPLES

The constructs described below are prepared using the general techniquesof genetic engineering and molecular cloning as detailed in Maniatis etal. (1989, Laboratory Manual, Cold Spring Harbor, Laboratory Press, ColdSpring Harbor, N.Y.) or in accordance with the manufacturer'srecommendations when a commercial kit is used. The homologousrecombination steps are preferably carried out in the E. coli strain BJ5183 (Hanahan, 1983, J. Mol. Biol. 166, 557-580). With regard torepairing the restriction sites, the technique employed consists inusing the large fragment (Klenow fragment) of E. coli DNA polymerase Ito fill in the 5′ protruding ends. In addition, the adenoviral genomefragments employed in the different constructs described below are shownprecisely in accordance with their positions in the nucleotide sequenceof the Ad5 genome, as disclosed in the Genebank database under referenceM73260.

With regard to the cell biology, the cells are transfected or transducedand cultured in accordance with the standard techniques which are wellknown to the skilled person. With regard to the yeast strains, use ismade of the conventional techniques which are described, in particular,in Guthrie and Fink (1991, Methods in Enzymology, Vol. 194, Guide toYeast Genetics and Molecular Biology, Academic Press Inc).

EXAMPLE 1

Constructing the Truncated FUR1Δ105 Gene

The EcoRI/HindIII fragment carrying the wild-type FUR1 gene is isolatedfrom the plasmid pHX (Kern et al., 1990, Gene 88, 149-157) and insertedinto the integrating yeast plasmid pRS306 (Sikorski and Hieter, 1989,Genetics 122, 19-27) which has been digested with these same enzymes, inorder to form the plasmid pRS306FUR1. Starting with this latter plasmid,the FUR1 gene is recloned, in the form of a XhoI/EcoRI fragment, intothe yeast centromeric plasmid pRS314 (Sikorski and Hierer, 1989,Genetics 122, 19-27), which has been treated with XhoI and EcoRI. Thisyields the phagemid pRS314FUR1, from which the uracil-containing singlestrand is prepared in the E. coli strain CJ236 (dut, ung, thi, relA,pCJ105 (Cm^(R))); site-directed mutagenesis is then carried out on thisstrand using the Kunkel technique (Muta-Gene phagemid kit, In vitroMutagenesis Version 2; Biorad). The aim of the mutagenesis is tointroduce EcoRI and MluI restriction sites 5′ of the initiating ATG and3′ of the stop codon, respectively, of the FUR1 gene and to modify thecontext of the initiating ATG in accordance with the Kozak rules, whichentails the second Asn codon being changed into Asp. Theoligonucleotides described in SID NO: 3 and NO: 4 are used to do this.The plasmid which is thereby generated, i.e. pRS314FUR1+1, is used as acontrol for the wild-type UPRTase activity.

In parallel, the first 105 nt of the FUR1 coding sequence are deleted bymutagenizing plasmid pRS314FUR1 using the oligonucleotides described inSID NO: 5 and NO: 4. The resulting plasmid, i.e. pRS314FUR1+106,contains the same modifications as its complete equivalent, namely anEcoRI site 5′ of the ATG codon (corresponding to the Met +36 of thenative protein), a Kozak consensus at this point, together with a changeof the following Ser codon into Ala, and an MluI site 3′ of the stopcodon. The EcoRI/SacI fragment from pRS314FURI+106 is recloned intoplasmid pRS314FUR1+1, which has been cleaved with EcoRI and SacI, inorder to form the plasmid pRS314FUR1Δ105, which carries an FUR1 genewhich expresses a protein whose first 35 N-terminal residues have beendeleted.

EXAMPLE 2

Complementation of an Furl Mutant of S. Cerevisiae

The functional ability of the FUR1 expression products obtained from theabove constructs is assessed by the complementation of an furl-8 trpl-4mutant of S. cerevisiae (Jund and Lacroute, 1970, J. Bacteriol. 102,607-615) which has been transformed with the different plasmids of thepRS314 type. The transformants are cultured either in the conventionalmanner (minimal YNB medium) or in the presence of 5 mM 5-FU, and theirability to grow at 30° C. and 37° C. is evaluated. The results arepresented in Table 1, where + represents growth and − represents theabsence of growth, testifying to sensitivity to 5-FU and therefore to aUPRTase activity.

TABLE 1 YNB medium (minimal) +5mM 5-FU Transformants 30° C. and 37° C.30° C. and 37° C. PRS314v[sic] + + pRS314FUR1 + − pRS314FUR1 + 1 + −pRS314FUR1 + 106 + − pRS314FUR1Δ105 + −

All the transformants are of course able to grow in conventional YNBmedium. By contrast, only the transformant harboring the “empty” pRS314plasmid, which does not contain the FUR1 sequences, is able to grow inthe presence of 5-FU whereas those transformants which have been giventhe plasmids containing the native FURL sequences (pRS314FUR1), the FUR1sequences which have been mutated at the level of the first ATG codon(pRS314FUR1+1), the FUR1 sequences which have been mutated at the levelof the second ATG codon (pRS314FUR1+106) or the truncated FUR1 sequences(pRS314FUR1Δ105) are sensitive to the presence of 5-FU. Thesecomplementation tests demonstrate that the different constructs producea functional UPRTase enzyme and that deleting the 35 N-terminal residuesdoes not have any negative effect on the activity of the enzyme.

EXAMPLE 3

Expressing FUR1 and FUR1Δ105 in Mammalian Cells

The EcoRI/MluI fragments of pRS314FUR1+105 and pRS314FUR1Δ105, carryingthe FUR1 and FUR1Δ105 genes, were introduced into the eukaryoticexpression plasmid pCI-neo (Promega) in order to form the plasmidspCI-neoFUR1+1 and pCI-neoFUR1Δ105, respectively. COS7 cells (ATCCCRL-1651) are transiently transfected with these plasmids and theUPRTase enzymic activity is determined in the cell extracts.

More precisely, 5×10⁵ COS7 cells are sown, at 37° C., in 60 mm culturedishes containing 5 ml of DMEM medium supplemented with 10% fetal calfserum (FCS). On the following day, the cells are treated with 20 μl oflipofectin (Gibco BRL) in the presence or absence of 5 μg of plasmid andculturing is continued in 2 ml of DMEM medium. After 16 hours ofincubation at 37° C., the cells are replaced in 5 ml of DMEM-10% FCS forapproximately 48 hours. The cells are then washed and resuspended in 30μl of lysis buffer (50 mM Tris-HCl, pH 7.5/150 mM NaCl/5 mM EDTA/1 mMDTT/1% triton X-100) at 4° C. for 30 minutes. The cell lysate iscollected and centrifuged, and the UPRTase enzymic activity is assayedon the resulting supernatant. To do this, 4 μl of lysate are incubatedat 37° C. for 10 minutes in the presence of 2 μl of UPTRase reactionbuffer (100 mM Tris-HCl pH 7.5/10 mM MgCl₂/10 mM 5-PRPP(5-phosphoribosylpyrophospate)/3 mM [C¹⁴]uracil having 0.02 μCi/μl(NEN)). The enzyme reactions are stopped by heating at 100° C. for oneminute. 1 μl aliquots are loaded onto a polyethyleneimine-cellulose TLCplate (Merck). The uracil is separated from the UMP by using water asthe migration solvent. Following migration, the TLC plate is scannedwith a PhosphorImager (445 SI; Molecular Dynamics). The proteinconcentration is measured by the Bradford method (protein Assay;Biorad). The UPRTase activity was determined on the basis of threeindependent transfections, and the results are shown in Table 2.

TABLE 2 UPRTase activity (37° C.) pmol of uracil transformed/Transfected plasmid minute/mg of protein — not detectable PCI-neo notdetectable PCI-neoFUR1 + 1 91 +/− 15 PCI-neoFUR1Δ105 3150 +/− 420

The transformation of uracil into UMP is only observed in the cellswhich have been transfected with the plasmids expressing a complete FUR1gene (pCI-neoFUR1+1) or truncated FUR1 gene (pCI-neoFUR1Δ105). It isalso noted that the UPRTase activity encoded by the truncated gene ishigher than that encoded by the complete gene by a factor of 30.

EXAMPLE 4

Constructing an Adenoviral Vector for Transferring the TruncatedFUR1Δ105 gene and infecting human tumor cells

1. Constructing pTG6289 for Expressing FUR1Δ105

The EcoRI/MluI fragment from pCI-neoFUR1Δ105, encompassing the truncatedgene, is isolated and introduced into the vector pTG6600, which has beencleaved with the same enzymes, in order to form the transfer vectorpTG6288. For information, pTG6600 is a p polyII vector (Lathe et al.,1987, Gene 57, 193-201) into which the Ad5 1 to 458 sequences, the earlyCMV promoter, the hybrid splicing sequences found in plasmid pCI(Promega Corp, comprising the splicing donor site of intron 1 of thehuman β-globin gene and the splicing acceptor site of the mouseimmunoglobulin gene), the SV40 virus polyadenylation sequences and theAd5 3328-5788 sequences have been inserted. Effecting such a constructis within the capacity of the skilled person, in particular on the basisof document FR-A-2 763 959. The adenoviral vector pTG6289 isreconstituted by recombination, in E. coli strain BJ 5183, between thePacI/BstEII fragment from pTG6288 and the ClaI-linearized vector pTG6624(described in document FR-A-2 763 959). For information, pTG6624corresponds to plasmid p poly II which is carrying the Ad5 genome, fromwhich the E1 (nt 459 to 3327) and E3 (nt 28592 to 30470) regions havebeen deleted, together with an expression cassette, which is inserted inplace of E1.

The final pTG6289 construct contains the Ad5 genome from which the bulkof the E1 (nt 459 to 3328) and E3 (nt 28249 to 30758) regions have beendeleted and, in place of E1, a cassette for expressing the FUR1Δ105gene, which is placed under control of the early CMV promoter and hybridβ-globin/Ig splicing sequences. The adenoviral particles are generatedby transfecting a cell line which complements the E1 function, forexample the 293 cell line (ATCC CRL1573) in accordance with thetechniques of the art (Graham and Prevec, 1991, Methods in MolecularBiology Vol. 7, Gene Transfer and Expression Protocols; Ed. E. J.Murray, The Human Press Inc, Clinton, N.J.).

2. Constructing a Vector for Expressing the FCY1 gene.

The uracil-containing single strand is isolated from the expressionphagemid pRS315FCY1 (Erbs et al., 1997, Curr. Genet. 31, 1-6), whichstrand is modified by means of site-directed mutagenesis (Muta-genePhagemid kit, In vitro Mutagenesis version 2; Biorad). The mutagenesisuses the oligonucleotides of SID NO: 6 and NO: 7, making it possible tointroduce a HindIII site 5′ of the initiating ATG, and an EcoRI site 3′of the stop codon, of the FCY1 gene and to modify the translationalcontext in accordance with the Kozak rules. The HindIII/EcoRI fragment,which has been modified in this way, is inserted into the plasmid pRS306(Sikorski and Hieter, 1989, Genetics 122, 19-27), which has beendigested with the same enzymes, in order to form pRS306FCY1. TheXhoI/XbaI fragment from pRS306FCY1 is subcloned into the vector pCI-neo(Promega), which has been cleaved with XhoI and XbaI. This results inpcI-neoFCY1 comprising the FCY1 gene having a Kozak consensus sequenceat the level of its intiating ATG.

An adenoviral vector for expressing the S. cerevisiae FCY1 gene,encoding the enzyme CDase, is generated. The XhoI/XbaI fragment fromplasmid pCI-neoFCY1, encompassing the FCY1 gene, is introduced into theXhoI-XbaI-digested vector pTG6600 in order to form pTG6286. As before,homologous recombination between the PacI/BstEII fragment, which carriesFCY1 and which was isolated from pTG6286, and the ClaI-linearized vectorpTG6624 generates the adenoviral vector pTG6287, which is deleted forthe E1 and E3 regions and which contains the FCY1 gene, instead of E1,placed under the control of the CMV promoter and hybrid β-globin/Igsplicing sequences. The viral particles (AdTG6287) are obtained bytransfecting 293 cells.

3. Determining the Enzyme Activities.

The adenoviral constructs are tested on three human tumor cell lines:PANC-1 (pancreatic carcinoma/ATCC CRL-1469), SK-BR-3 (breastadenocarcinoma/ATCC HTB-30) and SW480 (colon adenocarcinoma/ATCCCCL-228). These cells were chosen because of their resistance to 5-FU,thereby making it possible to determine the efficacy of the FUR1Δ105gene.

More precisely, 5×10⁶ cells suspended in 100 μl of PBS-2% FCS areincubated at 37° C. for 30 minutes in the presence of the adenovirusesto be tested (Ad.CMV-FCY1 and Ad.CMV-FUR1, or a mixture of the two) atan MOI (multiplicity of infection) of 20. As a negative control, eithera mock infection is carried out or an infection is carried out with afirst-generation adenovirus which lacks the gene of interest (Ad null).The infected cells are taken up in 500 μl of DMEM-10% FCS and placed ina 35 mm culture dish at a rate of 1×10⁶ cells per dish. After 48 hoursof incubation at 37° C., the different enzymic activities, using 5-FC or5-FU as a substrate, are determined on the cell lysates. The UPRTaseactivity is measured, in accordance with the abovedescribed protocol, on4 μl of cell lysate, which are incubated at 37° C. for 20 minutes in thepresence of 2 μl of UPRTase reaction buffer ([C¹⁴]5-FU, Sigma). In thecase of the CDase activity, use is made of 4 μl of cell lysate, whichare incubated at 37° C. for 20 min together with 2 μl of CDase reactionbuffer (100 mM Tris-HCl, pH 7.5/3 mM [H³]5-FC, having 0.25 μCi/μl,Sigma). The conversion of 5-FC into 5-FUMP (CDase/UPRTase activities) isdetermined on 4 μl of cell lysate which are incubated at 37° C. for 20min in the presence of 2 μl of CDase/UPRTase reaction buffer (100 mMTris-HCl, PH 7.5/10 mM MgCl₂/10 mM 5-PRPP/3 mM [H³]5-FC having 0.25μCi/μl). 1 μl aliquots are loaded onto a PEI cellulose TLC plate. The5-FU is separated from the 5-FC by using water as the solvent, and the5-FUMP is separated from the 5-FU by using a water/1-butanol (14%/86%)mixture as the solvent.

In the three cell types, CDase activity is observed in the extractsobtained from the cells which were infected with the adeiiovirusexpressing the FCY1 gene (Ad.CMV-FCY1) and from the cells which werecoinfected with the two viruses, i.e. Ad.CMV-FCY1 and Ad.CMV-FUR1Δ105.The enzyme assay shows a varying activity of from about 80 to 100 pmolof 5-FC transformed/min/mg of protein in the PANC-1, SK-BR3 and SW480cells. As far as the transformation of the 5-FU is concerned (UPRTaseactivity), an endogenous activity of approximately some one hundred pmolof 5-FU transformed/min/mg of protein is observed. The activity isamplified by a factor of 7 to 8 when the infection is carried out withthe Ad.CMV-FUR1Δ105 adenovirus on its own or when the cells arecoinfected. Finally, the conversion of 5-FC to 5-FUMP is detected in theextracts which have been infected with Ad-CMV-FCY1 (due to theendogenous UPRTase activity) and when the cells are coinfected.

In addition, the LD₅₀ (50% lethal dose) values of 5-FC and 5-FU (μM) aredetermined with regard to the three human tumor cell lines which areinfected with the above viruses (Mock infection, Ad null infection,Ad.CMV-FCY1 infection and Ad.CMV-FUR1Δ105 infection) or which arecoinfected with the adenoviruses expressing the FCY1 and FUR1Δ105 genes.The cells are infected at an MOI of 20 and then cultured in the presenceor absence of drugs (5-FC or 5-FU) at various concentrations. Trypanblue is added after trypsinizing on D+6. The living cells do not take uptrypan blue while the dead cells are stained. The % mortality isassessed under the microscope. The values reported below are the meansobtained after four counts.

The LD₅₀ of the infected cell lines with regard to 5-FC is between 5 and10 μM (depending on the cell lines) when the two FCY1 and FUR1+Δ105genes are coexpressed, whereas it is from 100 to 500 μM when the FCY1gene is expressed on its own and from 5000 to 10,000 μM in the case ofthe negative controls (Mock infection and Ad null infection) and in thecase of Ad.CMV-FUR1Δ105. In the same way, the LD₅₀ with regard to 5-FUis considerably lower when the infection uses the virus expressing theFUR1Δ105 gene as compared with the negative controls and in theinfection using Ad.CMV-FCY1 on its own. These results demonstrate theimportance of using the synergistic effect of the FCY1 and FUR1Δ105genes in a “suicide” gene approach for improving conversion of the 5-FCprodrug into cytotoxic 5-FUMP.

EXAMPLE 5

Constructing a Hybrid Gene which Encodes a Fusion Protein Carrying theCDase and UPRTase Activities

1. Constructing the Gene Fusion

The EcoRI/NotI fragment from pCI-neoFUR1Δ105 is inserted into theEcoRI/NotI-cut plasmid pCI-neoFCY1 in order to give the plasmidpCI-neoFCY1+FUR1Δ105. Using this uracil-containing single-strandedplasmid as the starting point, the FCY1 and FUR1Δ105are fused by meansof site-directed mutagenesis using the oligonucleotide oTG 11615 (SIDNO: 8). The modification consists in deleting the stop codon of FCY1 andthe initiating Met codon of FUR1, thereby enabling the FCY1 and FUR1Δ105sequences to be fused in-phase without any spacer (this fusion isdesignated FCU1 below).

2. Enzymic Characterization of the Bifunctional FCU1 Protein

The previously described techniques are used to determine the CDase andUPRTase activities (10 minute reaction at 37° C.) in cell lysates fromCOS7 cells which were:

untransfected

transfected with 10 μg of pCI-neo (empty plasmid)

transfected with 5 μg of pCI-neoFCY1+5 μg of pCI-neoFUR1Δ105

transfected with 5 μg of pCI-neo+5 μg of pCI-neoFCU1

The values obtained are listed in Table 3 below and are the means ofthree enzyme determinations.

TABLE 3 CDase UPRTase CDase/UPRTase pmol of cytosine pmol of uracil pmolof UMP transformed/min/ transformed/min/ formed/min/mg COS7 mg ofprotein mg of protein of protein Untrans- not detectable not detectablenot fected detectable pCI-neo not detectable not detectable notdetectable PCI- 626 +/− 106 3005 +/− 298 535 +/− 54 neoFCY1 + PCI-neoFUR1Δ105 pCI-neo + 5373 +/− 268  2789 +/− 310 2121 +/− 107pCI-neoFCU1

An equivalent UPRTase activity is observed whether the gene is in thefused form (pCI-neoFCU1) or in the unfused form (pCI-neoFUR1Δ105).Surprisingly, the CDase activity is increased by a factor of 10 when thegene is in the fused form as compared with the unfused FCY1 gene. Thus,the fused form is beneficial since it gives rise to an increase in theaggregate CDase-UPRTase activity and therefore to an increase in theformation of 5-FUMP from 5-FC.

The UPRTase activity of the fusion protein encoded by FCU1 is limitingin relation to the CDase activity; the formation of a pool of uracil ofthe order of 3000 pmol of uracil/min/mg of protein is detected in vitroduring the CDase-UPRTase reaction. This pool of uracil does not existunder the same conditions when the reaction makes use of the FCY1 andFUR1Δ105 proteins.

3. Expressing the FCY1, FUR1Δ105 and FCU1 genes, which have been clonedinto the plasmid pCI-neo, in B16(F0) Cell Lines

The efficiency of the FCU1 gene fusion is assessed by transfecting theB16(F0) mouse melanoma cell line (ATCC CRL-6322). In order to do this,2.5×10⁵ B16(F0) cells are seeded, at 37° C., in 60 mm dishes containing5 ml of DMEM-10% FCS medium. After 48 hours of incubation, the cellswere treated with 20 μl of lipofectin (Gibco BRL) in the presence of 5μg of plasmid and culturing was then continued at 37° C. for 16 hours in2 ml of DMEM medium. 48 hours after that, the cells are placed in 5 mlof DMEM-10% FCS and then diluted, in 10 cm Petri dishes, in DMEM-10% FCSmedium containing 1 mg of G418/ml. Clones which are resistant to G418are visible after 20 days. The clones obtained for each construct arecollected, transferred into T150 flasks and cultured in selectivemedium. The CDase, UPRTase and CDase-UPRTase activities are determinedat 37° C. in the presence of 5-FC or 5-FU as the substrate.

The values shown are the means obtained after three enzymedeterminations (see Table 4).

TABLE 4 CDase UPRTase CDase/UPRTase pmol of 5-FC pmol of 5-FU pmol of5-FUMP transformed/min/ tranformed/min/ formed/min/mg of B16 (F0) mg ofprotein mg of protein protein Untrans- not detectable 310 +/− 77 notdetectable fected pCI-neo not detectable 238 +/− 56 not detectablepCI-neoFCY1   3 +/− 0.4 292 +/− 93   2 +/− 0.4 pCI- not detectable 813+/− 47 not detectable neoFUR1Δ105 pCI-neoFCU1 158 +/− 9  834 +/− 67 41+/− 5 

The enzyme results confirm those determined using transfected COS7cells, namely a conserved UPRTase activity but a CDase activity which isstrongly increased in the case of the fusion protein as compared withthat measured using the native FCY1 protein. In the case of the B16clones transfected with pCI-neoFCU1 , a pool of 5-FU (120 pmol/min/mg ofprotein), which is the consequence of the increase in the CDaseactivity, also appears in vitro.

The sensitivities of the different clones to 5-FC and 5-FU are alsotested after incubating in the presence of the drugs for 6 days. Thefollowing values are the means of four counts (using trypan blue) ofcell mortality. The LD₅₀ for 5-FC is 10 μM in the case of the cellstransfected with the plasmid carrying the (pCI-neoFCU1) gene fusion ascompared with 100 μM when using pCI-neoFCY1 and 1000 μM when using thenegative controls (untransfected cells and cells transfected withpCI-neo) and when using pCI-neoFUR1Δ105. The LD₅₀ for 5-FU is 0.03 μMwhen the transfection is carried out using pCI-neoFUR1Δ105 orpCI-neoFCU1 as against 0.05 μM in all the other cases.

These results demonstrate that the expression of the FCU1 genesensitizes the cells to 5-FC more strongly than does the expression ofthe FCY1 gene. This difference in sensitization to 5-FC is due to thefusion protein having a higher CDase activity and not to the presence ofthe UPRTase activity; this UPRTase activity, which is encoded by the S.cerevisiae gene, has little effect on the toxicity of the 5-FU for theB16(F0) cells, which are naturally very sensitive to 5-FU and whichexhibit substantial endogenous UPRTase activity.

4. Demonstrating that B16(F0) cells which are transfected with the FCU1gene exhibit a bystander effect.

The appearance of a pool of 5-FU in the in vitro assay of theCDase-UPRTase activity of the FCU1-transfected B16(F0) cells is, apriori, an advantage for potentiating the bystander effect: the greaterthe size of this intracellular pool, the greater will be the passivediffusion of the 5-FU in the medium and the greater will be themortality of the untransfected cells.

In order to demonstrate this bystander effect, untransfected B16(F0)clones were mixed, in different proportions, with B16(F0) clones whichwere transfected with pCI-neoFCU1. These clone mixtures were then testedin the presence of different concentrations of 5-FC. Cell mortality(using trypan blue) is assessed after culturing for 4 days in thepresence of 5-FC. The results (means of 4 determinations) are presentedin Table 5 below.

TABLE 5 B16/B16 pCI-neoFCU1 LD₅₀ (μM) in % +5-FC 100%/0%  5000  95%/5% 500 90%/10% 300 75%/25% 200 50%/50% 100  0%/100%  50

It is seen that the LD₅₀ for 5-FC is lowered by a factor of 10 as soonas the cell mixture contains 5% of FCU1-transfected cells, therebytestifying to the existence of a bystander effect. The LD₅₀ does ofcourse continue to decrease when the proportion of transfected cellsincreases.

With the aim of demonstrating that this bystander effect does not,contrary to the tk/ganciclovir system, require any contact between thecells, due to the diffusion of the 5-FU into the medium, (untransfected)B16 cells were incubated for 4 days in medium (diluted one to four innew medium) which was derived from the culture supernatants from B16(negative control) or B16 pCI-neoFCU1 clones which have been incubatedfor 48 hours in the presence of different concentrations of 5-FC, andthe mortality of the B16 cells was assessed using trypan blue. Theresults demonstrate a drastic effect on the LD₅₀ and show that 5-FU ispresent in the medium as the consequence of the protein fusion encodedby the FCU1 gene having a high CDase activity.

EXAMPLE 6

Constructing an Adenoviral Vector for Transferring the FCU1 Gene andInfecting Tumor Cells.

1. Constructing pTG13060 for Expressing FCU1

The XhoI/MluI fragment from pCI-neoFCU1 , encompassing the FCU1 genefusion, is isolated and introduced into the XhoI/MluI-linearized vectorpTG6600 in order to form the transfer vector pTG13059. Homologousrecombination between the PacI/BstEII fragment, which carries FCU1 andwhich is isolated from pTG13059, and the ClaI-linearized vector pTG6624generates the adenoviral vector pTG13060, which is deleted for the E1and E3 regions and which contains, in the place of E1, the FCU1 geneplaced under the control of the CMV promoter and hybrid β-globin/Igsplicing sequences. The viral particles (Ad.CMV-FCU1) are obtained bytransfecting 293 cells.

2. Expressing the FCU1 Gene, which is Cloned in an Adenoviral Vector, inTumor Cells

The new adenoviral construct is tested on three human tumor cell lines:PANC-1, SK-BR-3 and SW480. The above-described techniques are used todetermine the CDase and UPRTase activities (employing 5-FC or 5-FU asthe substrate) in cell lysates obtained from the human cells, which wereinfected at an MOI of 20. A UPRTase activity (of from 700 to 800 pmol of5-FU transformed/min/mg of protein) is observed in the three cell linesinfected with Ad.CMV-FCU1 which is equivalent to the UPRTase activityexpressed by the FUR1Δ105 gene. In the three cell types, the CDaseactivity is amplified by a factor of 100 (10,000 to 12,000 pmol of 5-FCtransform/min/mg of protein) when the gene is expressed in fusion form(FCU1) as compared with the activity of the cells when infected withAd.CMV-FCY1.

When expressed from an adenoviral vector, the fusion protein exhibits aUPRTase activity, as in Example 5, which is limiting in relation to theCDase activity and which leads to the formation of a substantial pool of5-FU of the order of 10,000 pmol of 5-FU/min/mg of protein during theCDase-UPRTase reaction. This accumulation of 5-FU potentiates thebystander effect.

The LD₅₀ values for 5-FC and 5-FU are determined vis-à-vis the threehuman tumor cell lines infected with the different viruses (Ad null,Ad.CMV-FCY1, Ad.CMV-FUR1Δ105 and Ad.CMV-FCU1) or coinfected with theadenoviruses expressing the FCY1 and FUR1Δ105; genes. The cells areinfected at an MOI of 5 and then cultured in the presence of differentconcentrations of 5-FC and 5-FU. After 6 days of culture, the viabilityof the cells is determined using trypan blue. The LD₅₀ values listed inTable 6 are the means of four counts.

TABLE 6 LD₅₀ (μM) LD₅₀ (μM) 5-FU 5-FC PANC-1 Mock 0.3 10,000 Ad null 0.310,000 Ad.CMV-FCY1 0.3 500 Ad.CMV-FUR1Δ105 0.01 10,000 Ad.CMV-FCY1 +0.01 20 Ad.CMV-FUR1Δ105 Ad.CMV-FCU1 0.01 0.5 SK-BR-3 Mock 0.1 5000 Adnull 0.1 5000 Ad.CMV-FCY1 0.1 300 Ad.CMV-FUR1Δ105 0.005 5000Ad.CMV-FCY1 + 0.005 10 Ad.CMV-FUR1Δ105 Ad.CMV-FCU1 0.005 0.03 SW480 Mock0.5 5000 Ad null 0.5 5000 Ad.CMV-FCY1 0.5 300 Ad.CMV-FUR1Δ105 0.03 5000Ad.CMV-FCY1 + 0.03 20 Ad.CMV-FUR1Δ105 Ad.CMV-FCU1 0.03 0.3

These results demonstrate that expression of the FCU1 gene from anadenoviral vector sensitizes the cells to 5-FC more strongly than doesthe coexpression of the FCY1 and FUR1Δ105 genes. This greatersensitivity to 5-FC results from the increase in the CDase activityencoded by the FCU1 gene fusion.

3. Demonstrating that Human Tumor Cells Infected with the DifferentAdenoviruses Exhibit a Bystander Effect

The human tumor cells PANC-1, SK-BR-3 and SW480 are infected (Ad null,Ad.CMV-FCY1, Ad.CMV-FUR1Δ105 and Ad.CMV-FCU1) at an MOI of 20. After 48hours of incubation at 37° C., the cells are rinsed twice with PBS,trypsinized and spread out, in different proportions, in the presence ofuninfected cells. After 6 days of culturing in the presence of 1 mM5-FC, cell mortality is assessed using trypan blue. The results (mean of4 determinations) show that, in the case of the three tumor strains, amixture of 10% of cells infected with Ad.CMV-FCY1 and 90% of uninfectedcells leads to 100% mortality. When the cells are infected withAd.CMV-FCU1, 1% of infected cells is sufficient to lead to 100%mortality. This experiment demonstrates the existence of a substantialbystander effect, which results from the increase in the pool of 5-FU inthe cells expressing the fusion protein.

With the aim of demonstrating the secretion of 5-FU into the medium bycells expressing FCY1 and FCU1 in the presence of 5-FC, 10⁶ PANC-1,SK-BR-3 and SW480 cells are infected (Ad null, Ad.CMV-FCY1,Ad.CMV-FUR1Δ105 and Ad.CMV-FCU1) at an MOI of 20. The cells are culturedin the presence of 1 mM [³H]5-FC, giving 0.25 μCi/μl. After 48 hours ofculture, 2 μl aliquots of culture medium are loaded onto a cellulose TLCplate. The 5-FU is separated from the 5-FC by using a water/1-butanol(14%/86%) mixture as the solvent. In the three cell types, approximately20% of the 5-FC is converted into 5-FU when the cells are infected withAd.CMV-FCY1. More than 90% of the 5-FC is transformed into 5-FU when thecells are infected with Ad.CMV-FCU1.

4. Comparing the Efficacy of the FCU1/5-FC and TK/GCV Systems

With the aim of comparing the efficiency of the FCU1/5-FC system inrelation to the TK/GCV system, an adenoviral vector is generated forexpressing the herpes simplex virus type 1 thymidine kinase gene (HSV-1TK). The EcoRI/XbaI fragment from pTG4043, encompassing the HIV-1 TKcDNA, is isolated and introduced into the EcoRI/XbaI-linearized vectorpTG6600, in order to form the transfer vector pTG6222. Homologousrecombination between the PacI/BstEII fragment, which carries HSV-1 TKand which is isolated from pTG6222, and the ClaI-linearized vectorpTG6624 generates the adenoviral vector pTG13019, which is deleted forthe E1 and E3 regions and which contains, in the place of E1, the HSV-1TK gene placed under the control of the CMV promoter and hybridβ-globin/Ig splicing sequences. The viral particles (Ad.CMV-TK) areobtained by transfecting 293 cells.

The human cells PANC-1, SK-BR-3 and SW480, and also the murine cellsB16(F0) and RENCA (renal carcinoma, Murphy et al. J. Natl Cancer Inst.,1973, 50(4): 1013-25) are infected with Ad null, Ad.CMV-FCU1 orAd.CMV-TK. The cells are cultured in the presence of differentconcentrations of 5-FC or GCV. After 10 days of culture, the viabilityof the cells is determined by counting with trypan blue. The therapeuticindex (Table 7) corresponds to the LD₅₀ of the uninfected cells (or LD₅₀of the cells infected with Ad null)/LD₅₀ of the cells infected withAd.CMV-FCU1 or with Ad.CMV-TK ratio. The therapeutic index of theFCU1/5-FC system is generally higher than that of the TK/GCV system,indicating that the suicide gene strategy based on the FCU1/5-FCcombination is more therapeutically efficaceous.

TABLE 7 FCU1/5-FC TK/GCV therapeutic index therapeutic index B16 (F0)MOI 50 30 10 RENCA MOI 5 100 100 SW480 MOI 1 300 100 PANC-1 MOI 1 1000150 SK-BR-3 MOI 1 3000 1000

5. In vivo Experiments

In order to assess the ability of the FCU1/5-FC and TK/GCV systems toinhibit the growth of tumors in vivo, 3×10⁵ B16(F0) cells are injectedsubcutaneously into 6 groups of n=10 immunocompetent B6D2 mice (=D0). Assoon as the tumors become palpable (D+8), a “vehicle” buffer (10 mMTris, pH 8, 1 mM MgCl₂) or the adenoviruses (see the legend to FIGS. 1and 2), which are resuspended in this same buffer, are injected at adose of 5×10⁸ IU by the intratumor route, on three occasions (D+8, D+9and D+10). From D+8 onwards, 1 ml of a 0.9% saline solution, or 1 ml ofa 1% solution of 5-FC or 1 ml of a C.1% solution of GCV is injectedintraperitoneally twice a day up to D+43. The results (FIG. 1 and FIG.2) show an increase in survival in the group injected with Ad. CMV-FCU1and treated with 5-FC as compared with the Ad.CMV-TK group which istreated with GCV and with the control groups. These results confirm thevalue of combining the FCU1 gene and the 5-FC prodrug, in particularwhen implementing an antineoplastic treatment.

EXAMPLE 7

Combining the FCU1 Suicide Gene and Genes Encoding Cytokines.

The combination of FCU1 and cytokines (IFNγ and IL-2) is tested in vivoin the murine tumor model in which B16(F0) cells are implanted inimmunocompetent B6D2 mice. The adenoviruses encoding the cytokines areAd.CMV-muIFNγ (AdTG13048), which expresses murine IFNγ, andAd.CMV-HuIL-2 (AdTG6624) which expresses human IL-2.

The vector pTG13048, for expressing murine IFNγ, was constructed in thefollowing manner: The SalI/NotI fragment from pTG8390, encompassing thecDNA for murine IFNγ (muIFNγ), is isolated and introduced into theXhoI/NotI-linearized vector pTG6600 in order to form the transfer vectorpTG13047. Homologous recombination between the PacI/BstEII fragment,which carries muIFNγ and which is isolated from pTG13047, and theClaI-linearized vector pTG6624 generates the adenoviral vector pTG13048,which is deleted for the E1 and E3 regions and which contains, in placeof E1, the muIFNγ gene placed under the control of the CMV promoter andhyrid β-globin/Ig splicing sequences. The viral particles(Ad.CMV-muIFNγ) are obtained by transfecting 293 cells. The pTG6624vector, expressing human IL-2, is described in the document FR-A-2 763959.

3×10⁵ B16(F0) cells are injected subcutaneously into 10 groups of 13immunocompetent B6D2 mice (=D0). As soon as the tumors become palpable(D=7), a “vehicle” buffer (10 mM Tris, pH 8, 1 mM MgCl₂) or theadenoviruses, or a combination of adenoviruses (see the legend to FIGS.3 and 4), which are resuspended in this same buffer, are injected at adose of 2×10⁸ IU per adenovirus (4×10⁸ IU in total), by the intratumorroute, on three occasions (D+7, D+8 and D+9). From D+7 onwards, 1 ml ofan 0.9% saline solution or 1 ml of a 1% solution of 5-FC are injectedintraperitoneally twice a day. The survival curves (FIG. 3 and FIG. 4)show that synergy exists between the suicide gene and the cytokine whichis included in the composition of the invention. This synergy isillustrated by a rate of survival which is higher in the case of theFCU1/cytokine combinations as compared with the approach which consistsin using an adenovirus encoding the suicide gene on its own or using acytokine on its own.

8 1 216 PRT Saccharomyces cerevisiae 1 Met Ala Ser Glu Pro Phe Lys AsnVal Tyr Leu Leu Pro Gln Thr Asn 1 5 10 15 Gln Leu Leu Gly Leu Tyr ThrIle Ile Arg Asn Lys Asn Thr Thr Arg 20 25 30 Pro Asp Phe Ile Phe Tyr SerAsp Arg Ile Ile Arg Leu Leu Val Glu 35 40 45 Glu Gly Leu Asn His Leu ProVal Gln Lys Gln Ile Val Glu Thr Asp 50 55 60 Thr Asn Glu Asn Phe Glu GlyVal Ser Phe Met Gly Lys Ile Cys Gly 65 70 75 80 Val Ser Ile Val Arg AlaGly Glu Ser Met Glu Gln Gly Leu Arg Asp 85 90 95 Cys Cys Arg Ser Val ArgIle Gly Lys Ile Leu Ile Gln Arg Asp Glu 100 105 110 Glu Thr Ala Leu ProLys Leu Phe Tyr Glu Lys Leu Pro Glu Asp Ile 115 120 125 Ser Glu Arg TyrVal Phe Leu Leu Asp Pro Met Leu Ala Thr Gly Gly 130 135 140 Ser Ala IleMet Ala Thr Glu Val Leu Ile Lys Arg Gly Val Lys Pro 145 150 155 160 GluArg Ile Tyr Phe Leu Asn Leu Ile Cys Ser Lys Glu Gly Ile Glu 165 170 175Lys Tyr His Ala Ala Phe Pro Glu Val Arg Ile Val Thr Gly Ala Leu 180 185190 Asp Arg Gly Leu Asp Glu Asn Lys Tyr Leu Val Pro Gly Leu Gly Asp 195200 205 Phe Gly Asp Arg Tyr Tyr Cys Val 210 215 2 373 PRT Saccharomycescerevisiae 2 Met Val Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly MetAsp 1 5 10 15 Ile Ala Tyr Glu Glu Ala Ala Leu Gly Tyr Lys Glu Gly GlyVal Pro 20 25 30 Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val LeuGly Arg 35 40 45 Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu HisGly Glu 50 55 60 Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys ValTyr Lys 65 70 75 80 Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp MetCys Thr Gly 85 90 95 Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Val GlyGlu Asn Val 100 105 110 Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln ThrArg Gly His Glu 115 120 125 Val Val Val Val Asp Asp Glu Arg Cys Lys LysIle Met Lys Gln Phe 130 135 140 Ile Asp Glu Arg Pro Gln Asp Trp Phe GluAsp Ile Gly Glu Ala Ser 145 150 155 160 Glu Pro Phe Lys Asn Val Tyr LeuLeu Pro Gln Thr Asn Gln Leu Leu 165 170 175 Gly Leu Tyr Thr Ile Ile ArgAsn Lys Asn Thr Thr Arg Pro Asp Phe 180 185 190 Ile Phe Tyr Ser Asp ArgIle Ile Arg Leu Leu Val Glu Glu Gly Leu 195 200 205 Asn His Leu Pro ValGln Lys Gln Ile Val Glu Thr Asp Thr Asn Glu 210 215 220 Asn Phe Glu GlyVal Ser Phe Met Gly Lys Ile Cys Gly Val Ser Ile 225 230 235 240 Val ArgAla Gly Glu Ser Met Glu Gln Gly Leu Arg Asp Cys Cys Arg 245 250 255 SerVal Arg Ile Gly Lys Ile Leu Ile Gln Arg Asp Glu Glu Thr Ala 260 265 270Leu Pro Lys Leu Phe Tyr Glu Lys Leu Pro Glu Asp Ile Ser Glu Arg 275 280285 Tyr Val Phe Leu Leu Asp Pro Met Leu Ala Thr Gly Gly Ser Ala Ile 290295 300 Met Ala Thr Glu Val Leu Ile Lys Arg Gly Val Lys Pro Glu Arg Ile305 310 315 320 Tyr Phe Leu Asn Leu Ile Cys Ser Lys Glu Gly Ile Glu LysTyr His 325 330 335 Ala Ala Phe Pro Glu Val Arg Ile Val Thr Gly Ala LeuAsp Arg Gly 340 345 350 Leu Asp Glu Asn Lys Tyr Leu Val Pro Gly Leu GlyAsp Phe Gly Asp 355 360 365 Arg Tyr Tyr Cys Val 370 3 25 DNASaccharomyces cerevisiae 3 cgggtccatg gttgaattcg aaatg 25 4 19 DNASaccharomyces cerevisiae 4 tggtgtacgc gtgtgattt 19 5 26 DNASaccharomyces cerevisiae 5 cgaagccatg gtttgcagaa ttctag 26 6 25 DNASaccharomyces cerevisiae 6 gttaaaagct tcataggcca tggtg 25 7 21 DNASaccharomyces cerevisiae 7 agtagagaat tcagcacgct g 21 8 26 DNASaccharomyces cerevisiae 8 atggttccga agcctcacca atatct 26

What is claimed is:
 1. An isolated nucleotide sequence encoding apolypeptide comprising the amino acid sequence of SEQ ID NO:
 1. 2. Anisolated nucleotide sequence encoding a polypeptide comprising the aminoacid sequence of SEQ ID NO:
 2. 3. A recombinant vector comprising anisolated nucleotide sequence encoding a polypeptide comprising the aminoacid sequence of SEQ ID NO: 1 or SEQ ID NO:2 placed under the control ofelements which are required for expression in a host cell.
 4. Therecombinant vector of claim 3, wherein said vector is selected from thegroup consisting of a plasmid vector and a viral vector, whereappropriate combined with one or more substances which improve thetransfectional efficacy or stability of the vector.
 5. The recombinantvector of claim 4, wherein said vector is a viral vector from a poxvirus, an adenovirus, a retrovirus, a herpes virus, an alphavirus, afoamyvirus, or an adenovirus-associated virus.
 6. The recombinant vectorof claim 4, wherein said vector is an adenoviral vector which lacks allor part of at least one region which is essential for replication andwherein the region is the E1, E2, E4, or L1-L5 region.
 7. The vectoraccording to claim 6, wherein said vector is an adenoviral vector whichadditionally lacks all or part of the non-essential E3 region.
 8. Therecombinant vector according to claim 3, wherein the elements which arerequired for expression comprise a promoter.
 9. A viral particlecomprising the recombinant vector of claim
 3. 10. A host cell comprisingan isolated nucleotide sequence encoding a polypeptide comprising theamino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2.
 11. A compositioncomprising an isolated nucleotide sequence encoding a polypeptidecomprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 and asecond nucleotide sequence of interest.
 12. The recombinant vectoraccording to claim 8, wherein the promoter is the cytomegalovirus (CMV)early promoter.